PROJECT SUMMARY Congenital heart disease (CHD) is a leading cause of death in newborns. Surgical intervention is the only effective treatment, but is limited by morbidity from available prosthetic vascular grafts. Tissue engineering offers a potential solution. We developed the first tissue engineered vascular graft (TEVG) for use in children. FDA approval has now been obtained for investigation of this device. Although TEVG is safe and effective, stenosis is the primary graft-related complication, representing a critical barrier for widespread clinical use. Preliminary mechanisms indicate that host macrophage infiltration is essential for vascular neotissue formation, however, excessive infiltration leads to stenosis. Macrophage infiltration is decreased by bone marrow-derived mononuclear cells (BM-MNC) that are seeded onto the scaffold as part of the implantation protocol. BM-MNC are transient and do not contribute to vascular tissue formation, but they do modulate the host inflammatory response through an unknown paracrine mechanism. Preliminary data demonstrate that IL-10 levels correlate positively with the number of BM-MNC and inversely with stenosis. Therefore, IL-10 represents a logical cytokine for this paracrine mechanism. The goal of this project is to determine how IL-10 modulates the host- TEVG interaction. We will determine if IL-10 is necessary for BM-MNC to modulate host macrophage function using an IL-10 null transgenic mouse BM-MNC donor compared to a wild-type BM-MNC donor. If BM-MNC IL-10 signaling is required, then removing IL-10 in donor animals will eliminate the ability of BM-MNC to inhibit stenosis. Next, we will determine if IL-10 is sufficient to modulate host macrophage function by administering systemic IL-10 and assessing the degree of macrophage infiltration. We hypothesize that systemic administration of IL-10 will decrease TEVG stenosis. We will identify the cellular source of IL-10 to determine if sorting BM-MNCs prior to seeding decreases stenosis. Lastly, we will determine the role of recipient IL-10 signaling on neovessel formation and stenosis. Successful completion of these aims will result in an improved mechanistic understanding of vascular neotissue formation, stenosis and how the host interacts with biomaterials. This approach will allow us to move away from empiric design and towards rational design of TEVG. This will contribute to the successful translation of TEVG into widespread clinical use and the improvement of outcomes for children with congenital cardiac anomalies.